The present invention pertains, in general, to methods for producing transgenic seeds. In particular, it relates to methods for ensuring that crop plants which are heterozygous for the presence of a transgene produce seeds which always carry the transgene.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
A major problem associated for the commercialization of transgenic cultivars in highly heterozygous crops is the segregation of transgenes during seed production (Conner and Christey Biocontrol Science and Technology 4:463-473 (1994)). In order to develop a cultivar involving crosses between heterozygous individuals (e.g.: asparagus, forage brassicas, pasture species, forest trees, etc.), it will be necessary to intermate individuals heterozygous for transgenes. In many open pollinated or synthetic cultivars this will usually involve the intermating of several transgenic lines independently derived for different individual plants. This will involve the parents of synthetic cultivars, or a sufficient number of different individuals to maintain an effective population size to avoid inbreeding depression/genetic drift within the population. The transgenic individuals utilized in producing transgenic synthetic populations may arise from single event transformations of a single plant. When this is the case, the transgene could be introduced into a synthetic population, such as an alfalfa synthetic population, by making multiple crosses of the individual transgenic alfalfa plant with a number of different non-transgenic alfalfa plants from one or more alfalfa lines. Alternatively, since the transgenic individuals to be intermated may be derived from independently derived transformed plants, the is transgenes may be located at different loci. The resulting intermated progeny will therefore be segregating at all the loci and the transgenic traits will have a xe2x80x9cquantitative basisxe2x80x9d (Conner and Christey, supra). As discussed immediately below, the prior art has failed to address the segregation and consequent loss of transgenes in open pollinated and synthetic populations.
U.S. Pat. No. 5,254,801 discloses methods whereby plant cells and whole plants can be genetically modified so as to selectively induce cellular lethality using heterologous dominant, conditionally lethal genes in combination with selected protoxin compounds. The methods are for inducing male sterility for the hybrid seed production, including alfalfa, canola, and oil seed rape. This patent fails to disclose a method of producing heterologous plants which utilizes a transgene coding for resistance to a specific phytotoxin.
U.S. Pat. No. 5,278,057 describes a method of producing plants with a marker closely linked to a target locus, in particular a nuclear male sterile target locus. The method involves transformation of a group of plants in order to introduce a marker into each plant, and isolation of a plant with the marker closely linked to a target locus. The markers include visible markers and dominant conditional lethal markers (e.g., antibiotic resistance or herbicide resistance). The method is of particular use for hybrid seed production of any crop plant where the target locus is a nuclear male sterile locus, including rapeseed, alfalfa, clover, cole crops or Brassica oleracea. 
U.S. Pat. No. 5,426,041 discloses a method for seed preparation which comprises:
a) crossing a male sterile plant and a second plant which is male fertile,
b) obtaining seed of said male sterile plant, wherein the seed has integrated into its genome:
1) a first recombinant DNA molecule having a first DNA sequence which encodes a first gene product and a first promoter which is capable of regulating the expression of said first DNA sequence; and,
2) a second recombinant DNA molecule which contains a second DNA sequence which encodes a second gene product and a second promoter which is capable of regulating the expression of said second DNA sequence.
The first and second gene products cooperate to selectively interfere with the function and/or development of cells of a plant that are critical to pollen formation and/or function of a plant grown from said seed, such that any plant grown from the seed is substantially male sterile.
More specifically, the ""041 patent further teaches a procedure to produce hybrid seed which includes using an IamS/IamH genetic system. The procedure can include linking the IamH gene to a gene for herbicide resistance so that the herbicide can be used for the roguing of the plant line A1; and, that herbicide application takes place after flowering and will kill the A1 so that only seed that has the genotype A1/A2 is produced. The A1/A2 seed is substantially 100% male sterile and can be pollinated with a male fertile line leading to commercial hybrid seed.
U.S. Pat. No. 5,633,441 is directed to plants comprising female-sterility-DNA encoding a protein or polypeptide such as barnase which, when produced in the cells of the plant, kills or significantly disturbs the metabolism, functioning or development of the cells. The foreign DNA also comprises a first promoter which directs expression of the female-sterility DNA selectively in style cells, stigma cells or style and stigma cells of the female reproductive organs of the plants. The first promoter does not direct detectable expression of the female sterility DNA in the ovule or in other parts of the plant so that the plant remains male-fertile. The female-sterility DNA is in the same transcriptional unit as and under the control of the first promoter. More specifically, the ""441 patent discloses a foreign chimaeric DNA sequence that comprises the female-sterility DNA and a first promoter and that can also comprise a marker DNA and a second promoter. Preferred markers include herbicide tolerance or resistance genes.
The ""441 patent further discloses a process for producing hybrid seeds, which grow into hybrid plants, by crossing: 1) the female-sterile plant of this invention which may include, in its nuclear genome, the marker DNA, preferably encoding a protein conferring a resistance to a herbicide on the plant; and 2) a female-fertile plant without the marker DNA in its genome.
U.S. Pat. No. 5,652,354 relates to promoters from endogenous genes of plants, is wherein said promoters direct gene expression selectively in stamen cells of said plant, particularly in tapetum cells of said plant. The promoters may be used to transform a plant with a foreign DNA sequence encoding a product which selectively disrupts the metabolism, functioning, and/or development of stamen cells of the plant. The male-sterility DNA and its associated promoter are exemplified as being foreign DNA sequences. Preferred marker DNAs are those which inhibit or neutralize the action of herbicides.
None of these patents discloses a method of producing an open-pollinated population or synthetic variety whereby a transgene is maintained at sufficiently useful levels during subsequent generations of inter- and intra-crossing of the parental lines which made up the original population or variety.
It would be highly desirable to have a method to prevent the formation of, or eliminate, the individual seeds that do not carry a transgene. If this could be achieved, all the seeds in subsequent generations would carry a transgene, without interfering with the highly heterozygous genetic background of the cultivar. It would also offer a more convenient strategy for introgression of transgenes into open pollinated crop cultivars. A single transgenic individual could be intermated to many other individuals, with the high proportion of non-transgenic progeny being prevented from developing in seed production blocks prior to or during flowering and seed development.
Thus, the object of this invention to provide methods for producing segregating populations in which one or more transgenes are maintained in a large enough percentage of the plants so that the beneficial effect of the transgenes are realized.
In one aspect, the present invention can be said to broadly consist in a method for biasing a crop plant which is heterozygous for a transgene towards the production of seeds which carry the transgene comprising the step of contacting a crop plant containing a gene construct comprising a transgene coding for resistance to a specific phytotoxin with said specific phytotoxin one or more times during the life of said plant.
In still another aspect the invention provides a method of selectively inhibiting phytotoxin-sensitive plant ovules, embryos and/or pollen in order to bias a crop plant which is heterozygous for a transgene towards the production of seeds which carry the transgene comprising the step of contacting a crop plant containing a gene construct comprising a transgene coding for resistance to a specific phytotoxin with said specific phytotoxin one or more times during the life of said plant.
In yet another aspect, the invention provides a method of selectively inhibiting phytotoxin-sensitive plant ovules in order to bias a crop plant which is heterozygous for a transgene towards the production of seeds which carry the transgene comprising the step of contacting a crop plant containing a gene construct comprising a transgene coding for resistance to a specific phytotoxin with said specific phytotoxin one or more times during the life of said plant.
In a further aspect, the invention provides a method of selectively aborting phytotoxin-sensitive plant embryos in order to bias a crop plant which is heterozygous for a transgene towards the production of seeds which carry the transgene comprising the step of contacting a gene construct comprising a transgene coding for resistance to a specific phytotoxin with said specific phytotoxin one or more times during the life of said plant.
In yet a further aspect, the invention provides a method of selectively inhibiting phytotoxin-sensitive pollen in order to bias a crop plant which is heterozygous for a transgene towards the production of seeds which carry the transgene comprising the step of contacting a crop plant containing a gene construct comprising a transgene coding for resistance to a specific phytotoxin with said specific phytotoxin one or more times during the life of said plant.
In addition to the phytotoxin resistance gene, the gene construct may also contain additional liked transgene(s) which are co-transferred to the transgenic plant.
Conveniently, the method includes the preliminary step of introducing said gene construct into said plant or into the seed from which said plant is grown.
In preferred embodiments, the method includes the subsequent step of collecting the seed produced by the plant, and confirming the presence of the gene construct.
The phytotoxin which is applied to the plant can be an antibiotic or a herbicide. It is however presently preferred that the phytotoxin be a herbicide. It is further preferred that the herbicide be one which translocates throughout the plant upon application.
In a further embodiment, the invention provides seeds carrying a gene construct produced by the method defined above.
Although the present invention is broadly as defined above, it will be appreciated by those person skilled in the art that it is not limited thereto and that it further includes the embodiments which are described below.
Further objects and advantages of the present invention will be clear from the description that follows.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
It will be appreciated from the above that the method of the invention has application to all crops where seed is produced from heterozygous plants. Such crop plants include forage crops such as forage brassica, forage legumes and grasses, trees, vegetables and ornamental flowers.
The critical step of the method is the application of a phytotoxin to the crop plant during its life. Usually, the phytotoxin will be applied during the vegetative or reproductive growth phase of the plant. The plant to which the phytotoxin is applied will be one which has either been directly transformed by introduction of a gene construct (comprising a gene coding for resistance to the phytotoxin and, optionally, one or more closely linked additional genes) or one which has been grown from seed which itself carries the gene construct.
It is contemplated that the phytotoxin be applied to the plant only once during its life. However, in preferred embodiments, the phytotoxin will be applied more than once, and most preferably two or three during the vegetative or reproductive growth phase.
As indicated above, the gene construct can be two components. The first component, which will always be present, is a gene coding for resistance against a phytotoxin. The phytotoxin resistance component of the gene construct can code for genes conferring resistance to antibiotics or similar chemicals e.g. kanamycin resistance (Bevan et al. Nature 304: 184-187 (1983); Fraley et al., Proceedings of the National Academy of Sciences USA 80: 4803-4807 (1983); Herrera-Estrella et al., The EMBO Journal 2: 987-995 (1983); De Block et al., The EMBO Journal, 3; 1681-1689 (1984); Horsch et al., Science, 223: 469-498 (1984); Horsch et al., Science, 227: 1229-1231 (1985)), methotrexate resistance (Herrera-Estrella et al., The EMBO Journal 2: 987-955 (1983)), chloramphenicol resistance De Block et al., The EMBO Journal, 3: 1681-1689 (1984)), hygromycin resistance (Walden et al., Plant Molecular Biology 5: 103-108 (1985); van der Elzen et al., Plant Molecular Biology 5: 299-302), bleomycin resistance (Hille et al., Plant Molecular Biology 7: 171-176 (1986), streptomycin resistance (Jones et al., Molecular and General Genetics 210: 86-91 (1987)), phleomycin resistance (Perez et al., Plant Molecular Biology 13: 365-373), sulfonamide resistance (Guerineau et al., Plant Molecular Biology 15: 127-136 (1990)), S-aminoethyl L-cysteine resistance (Perl et al., Bio Technology 11: 715-718 (1993), and genetamycin resistance (Hayford et al., Plant Physiology 86: 1216-1222 (1988); Gossele et al., Plant Molecular Biology 26: 2009-2012 (1994)).
It is however preferred that it be a coding for herbicide resistance, such as glyphosate resistance (Comai et al., Nature 317: 741-744 (1985)), phosphinothricin resistance (De Block et al., The EMBO Journal 6: 2513-2518 (1987); Wohllenben et al., Gene 70: 25-37 (1988)), atrazine resistance (Cheung et al., Proceedings of the National Academy of Sciences USA 85: 391-395 (1988)); sulfonylurea resistance (Haughn et al., Molecular and General Genetics 210: 266-271 (1988); Lee et al., The EMBO Journal, 7: 1241-1248 (1988)), bromoxynil resistance (Stalker et al., Science 242: 419-423 (1988)), 2,4-D resistance (Steber and Willmitzer Bio/Technology 7: 811-816 (1989); Lyon et al., Plant Molecular Biology 13: 533-540 (1989); Bayley et al, Theoretical and Applied Genetics 83: 645-649 (1992)), cyanamide resistance (Maier-Greniner et al., Angewandte Chemie, International Edition in English 30: 1314-1315 (1991)), and dalapon resistance (Buchanan-Wollaston et al., Plant Cell Reports 11: 627-631 (1992)).
It is especially preferred that this component of the construct code for resistance against a herbicide which is capable of translocating throughout the plant to which it is applied. Examples of such herbicides are chlosulfuron and N-phosphonomethylglycine (glyphosate).
The second, and optional, component of the gene construct is at least one additional gene directly or closely linked to the phytotoxin resistance gene. This gene can be any transgene (i.e. a gene from another plant or organism or a synthetic gene) desirable for introduction into the particular crop plant. Examples of such transgenes include resistance to pests and diseases e.g. insect-resistance cotton and corn (Perlak et al., Bio/Technology 8: 939-943 (1990); Koziel et al., Bio/Techhnology 11: 194-200)), virus-resistant alfalfa (Hill et al., Bio/Techhnology 9: 373-377 (1991)) and fungal-resistant rapeseed (Broglie et al., Science 254: 1194-1197 (1991)) or improved quality traits such as improved protein content in alfalfa (Schroeder et al., Australian Journal of Plant Physiology 18: 495-505 (1991) and modified oils in rapeseed (Knutson et al., Proceedings of the National Academy of Sciences USA 89: 2624-2628 (1992)). Alternatively the second component may not be a transgene but instead be an existing gene within the chromosomes of the plant which it is desirable to introduce an extra copy of.
The construct can be introduced into the plant or seed using any suitable procedure known in the art. Examples of such procedures include Agrobacterium-mediated transformation, direct DNA transfer to plant protoplasts or intact plant tissues using techniques such as electroporation, chemical-induced uptake, or microprojectile bombardment, or any of the range of other methods that are reported to accomplish gene transfer (seer reviews by Gasser and Fraley, Science 244: 1293-1299 (1989); Potrykus, Annual Review of plant Physiology and Plant Molecular Biology 42: 205-225 (1991); Klein et al., Bio/Techhnology 10: 286-291 (1992) and citations within these reviews).
It will be appreciated that when the gene construct includes the phytotoxin resistance gene only, it can nevertheless be linked to a gene of the plant which it would be desirable to ensure is always present in the seed obtained from the plant. This can be achieved by introducing the gene construct containing the phytotoxin resistance gene into the plant at an integration site very close to the site of the gene in question.
The seed which carries the gene construct is of course the commercial focus of the invention. The presence of the construct can be determined directly in the seed itself using methods routinely available in this art (for example using nucleic acid hybridization protocols) or can be demonstrated in the plant which is grown from the seed.
As used herein, the term xe2x80x9calfalfaxe2x80x9d means any Medicago species, including, but not limited to, M. sativa, M. murex, M. falcata, and M. prostrata. Thus, as used herein, the term xe2x80x9calfalfaxe2x80x9d means any type of alfalfa including, but is not limited to, any alfalfa commonly referred to as cultivated alfalfa, diploid alfalfa, glanded alfalfa, purple-flowered alfalfa, sickle alfalfa, variegated alfalfa, wild alfalfa, or yellow-flowered alfalfa.
As used herein, the term xe2x80x9callelexe2x80x9d means any of several alternative forms of a gene.
As used herein, the term xe2x80x9ccloverxe2x80x9d means means any Trifolium species, including, but not limited to, T. hybridum, T. vesiculosum, T. alexandrinum, T. incarnatum, T. campestre, T. dubium, T. ambiguum, T. arvense, T. pratense, T. fragiferum, T. subterraneum, T. repens, and T. medium. Thus, as used herein, the term xe2x80x9ccloverxe2x80x9d means any type of clover including, but is not limited to, any clover commonly referred to as alsike clover, arrowleaf clover, berseem clover, crimson clover, large hop clover, small hop clover, Kura clover, rabbit""s foot clover, red clover, strawberry clover, subterranean clover, white clover, and zigzag clover.
As used herein, the term xe2x80x9ccrop plantxe2x80x9d means any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food additives, smoking products, pulp production and wood production.
As used herein, the term xe2x80x9ccross pollinationxe2x80x9d or xe2x80x9ccross-breedingxe2x80x9d means the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of a flower on another plant.
As used herein, the term xe2x80x9ccultivarxe2x80x9d means a variety, strain or race of plant which has been produced by horticultural or agronomic techniques and is not normally found in wild populations.
As used herein, the term xe2x80x9cgenotypexe2x80x9d means the genetic makeup of an individual cell, cell culture, plant, or group of plants.
As used herein, the term xe2x80x9cheterozygotexe2x80x9d means a diploid or polyploid individual cell or plant having different alleles (forms of a given gene) at least at one locus.
As used herein, the term xe2x80x9cheterozygousxe2x80x9d means the presence of different alleles (forms of a given gene) at a particular gene locus.
As used herein, the term xe2x80x9chomozygotexe2x80x9d means an individual cell or plant having the same alleles at one or more loci.
As used herein, the term xe2x80x9chomozygousxe2x80x9d means the presence of identical alleles at one or more loci in homologous chromosomal segments.
As used herein, the term xe2x80x9chybridxe2x80x9d means any individual plant resulting from a cross between parents that differ in one or more genes.
As used herein, the term xe2x80x9cinbredxe2x80x9d or xe2x80x9cinbred linexe2x80x9d means a relatively true-breeding strain.
As used herein, the term xe2x80x9clocusxe2x80x9d (plural: xe2x80x9clocixe2x80x9d) means any site that has been defined genetically. A locus may be a gene, or part of a gene, or a DNA sequence that has some regulatory role, and may be occupied by different sequences.
As used herein, the term xe2x80x9cmass selectionxe2x80x9d means a form of selection in which individual plants are selected and the next generation propagated from the aggregate of their seeds.
As used herein, the term xe2x80x9copen pollinationxe2x80x9d means a plant population that is freely exposed to some gene flow, as opposed to a closed one in which there is an effective barrier to gene flow.
As used herein, the terms xe2x80x9copen-pollinated populationxe2x80x9d or xe2x80x9copen-pollinated varietyxe2x80x9d mean plants normally capable of at least some cross-fertilization, selected to a standard, that may show variation but that also have one or more genotypic or phenotypic characteristics by which the population or the variety can be differentiated from others. A hybrid which has no barriers to cross-pollination is an open-pollinated population or an open-pollinated variety.
As used herein, the term xe2x80x9covulexe2x80x9d means the female gametophyte, whereas the term xe2x80x9cpollenxe2x80x9d means the male gametophyte.
As used herein, the term xe2x80x9cphenotypexe2x80x9d means the observable characters of an individual cell, cell culture, plant, or group of plants which results from the interaction between that individual""s genetic makeup (i.e., genotype) and the environment.
As used herein, the term xe2x80x9cself-incompatiblexe2x80x9d means the failure, following mating or pollination, of a male gamete and a female gamete to achieve fertilization, where each of them is capable of uniting with other gametes of the breeding group after similar mating or pollination (Mather, J. Genet. 25:215-235 (1943)).
As used herein, the term xe2x80x9cself pollinatedxe2x80x9d or xe2x80x9cself-pollinationxe2x80x9d means the pollen of one flower on one plant is applied (artificially or naturally) to the ovule (stigma) of the same or a different flower on the same plant.
As used herein, the term xe2x80x9csyntheticxe2x80x9d means a set of progenies derived by intercrossing a specific set of clones or seed-propagated lines. A synthetic may contain mixtures of seed resulting from cross-, self-, and sib-fertilization.
As used herein, the term xe2x80x9ctransformationxe2x80x9d means the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term xe2x80x9cgenetic transformationxe2x80x9d means the transfer and incorporation of DNA, especially recombinant DNA, into a cell.
As used herein, the term xe2x80x9ctransgenicxe2x80x9d means cells, cell cultures, plants, and progeny of plants which have received a foreign or modified gene by one of the various methods of transformation, wherein the foreign or modified gene is from the same or different species than the species of the plant receiving the foreign or modified gene.
As used herein, the term xe2x80x9cvarietyxe2x80x9d means a subdivision of a species, consisting of a group of individuals within the species which are distinct in form or function from other similar arrays of individuals.
1. Alfalfa
Alfalfa (Medicago sativa L.) is an important forage species for hay and pasture which has been referred to as the xe2x80x9cQueen of the Foragesxe2x80x9d because of its high yields and feeding value. Alfalfa is recognized as the most widely adapted agronomic crop, as an effective source of biological nitrogen (N2) fixation, useful in the improvement of soil tilth, as an important source of protein yield/ha, and as an attractive source of nectar for honey bees. For a comprehensive review of the benefits of alfalfa as an agronomic crop, see Barnes et al., Highlights in the USA and Canada 1:2-24, In Alfalfa and Alfalfa Improvement, Hanson et al. (ed.), American Society of Agronomy, Monograph No. 29 (1988).
Although alfalfa originated in southwestern Asia, it is well adapted to a wide range of climates and soils in the United States, where about 11 million ha are grown annually. Between 1900 and 1975 more than 160 cultivars were developed for production in North America. Most of the newer cultivars were selected for improved adaptation and multiple pest resistance. For a comprehensive review of the distribution, history and origin of alfalfa, see Michaud et al, World Distribution and Historical Development 2:25-91, In Alfalfa and Alfalfa Improvement, supra; and, Quiros et al., The Genus Medicago and the Origin of the Medicago sativa Complex 3:93-124, In Alfalfa and Alfalfa Improvement, supra.
The genus Medicago is widely distributed and comprises an array of diverse species that are either annual or perennial. The most recent taxonomic studies of the perennial species concluded that M. sativa is polymorphic. Lesins and Gillies (Taxonomy and cytogenetics of Medicago 353-386, In Alfalfa science and technology, C. H. Hanson (ed.), American Society of Agronomy, (1972)) defined the complex as M. sativa-falcata-glutinosa, and Gunn et al. (USDA Tech. Bull. No. 1574 (1978)) designated it as the M. sativa sensu lato complex.
M. sativa plants are autopolyploid organisms, or more specifically, autotetraploids. More specifically, M. sativa plants are polysomic polyploid organisms which display tetrasomic inheritance patterns.
Essentially all annual species are cleistogamous and are exclusively self-pollinated. Generally, the perennial species require tripping, as by insect visits to the floral structures, and will set seed from either self or cross-pollination. Crosses can be made among subspecies in the M. sativa complexes and between the cultivated tetraploids and wild diploids without special preparation of the parents. For a comprehensive review of the floral characteristics, plant culture, and methods of self-pollinating or hybridizing alfalfa, see D. K. Barnes, Alfalfa 9:177-187, In Hybridization of Crop Plants, Fehr et al. (ed.), American Society of Agronomy Inc. (1980).
Commercial alfalfa seed may be provided either in a synthetic variety or a hybrid variety. Commercial production of synthetic varieties may include a breeder seed production stage, a foundation seed production stage, a register seed production stage and a certified seed production stage. Hybrid variety seed production may involve up to three stages including a breeder seed production stage, a foundation seed production stage and a certified seed production stage.
Breeder seed is an initial increase of seed produced from the strains or clones that are developed by a breeder. Foundation seed is a second generation increase of seed produced from the breeder seed. Register seed may be derived from foundation seed. Certified seed may be derived from breeder seed, foundation seed or register seed. Breeder seed descends from a selection of recorded origin, under the direct control of the breeder, a delegated representative or a state or federal inspection service, such as the AOSCA (Association of Official Seed Certification Analysts) in the U.S.A. Certified seed is used in commercial crop production. Certified seed is usually grown, processed and labeled under supervision and regulation of a public agency.
Efforts in developing healthy and productive alfalfa varieties often focus on breeding for disease and stress-resistant cultivars, for example, breeding for persistence, breeding for adaptation to specific environments, breeding for yield per se, and breeding for quality. Success has been attained in breeding for resistance to fungal, bacterial, insect, and nematode pests, including, but not limited to the development of varieties tolerant/resistant to bacterial wilt and common leaf spot (see, e.g., Elgin, Jr., et al., Breeding for Disease and Nematode Resistance 827-858, In Alfalfa and Alfalfa Improvement, supra) and to the spotted alfalfa aphid and alfalfa weevil (see, e.g., Sorensen et al., Breeding for Insect Resistance 859-902, In Alfalfa and Alfalfa Improvement, supra). Breeders have had less success in breeding for yield and quality per se (see, e.g. Hill et al., Breeding for Yield and Quality 26:809-825, In Alfalfa and Alfalfa Improvement, supra), although methods have been developed that help increase productivity and yield (U.S. Pat. No. 4,045,912). Historically, yield and productivity, quality and persistence are objectives of high concern to farmers.
2. Clover
The clover species described herein are the true clovers belonging to the genus Trifolium. The species of major agricultural importance in the Untied States are red clover (T. pratense L.), white clover (T. repens L.), crimson clover (T. incarnatum L.), and alsike clover (T. hybridum L.). Crimson clover is an annual species, and the others are perennial. These clovers are thought to have originated in Asia minor or southeastern Europe. They are grown extensively for hay, pasture, and soil improvement throughout the eastern half of the United States and under irrigation in the Pacific and adjacent states.
The genus Trifolium consists of about 240 species divided by taxonomists into about 16 sections (Hossain, Notes R. Bot. Gard. Edinb. 23:387-481(1961)). For the most part, gene transfer between sections by conventional hybridization methods has been difficult. About 30% of the clover species are self-incompatible and are cross-pollinated by bees, and 70% are self-pollinated. Some species (e.g., crimson clover) normally cross-pollinate, but set considerable seed upon selfing. For a comprehensive review of the floral characteristics, plant culture, and methods of self-pollinating or hybridizing clover, see N. L. Taylor, Clovers 16:261-272, In Hybridization of Crop Plants, supra.
1. Open-Pollinated Populations
The improvement of open-pollinated populations of such crops as rye, many maizes and sugar beets, herbage grasses, legumes such as alfalfa and clover, and tropical tree crops such as cacao, coconuts, oil palm and some rubber, depends essentially upon changing gene-frequencies towards fixation of favorable alleles while maintaining a high (but far from maximal) degree of heterozygosity. Uniformity in such populations is impossible and trueness-to-type in an open-pollinated variety is a statistical feature of the population as a whole, not a characteristic of individual plants. Thus, the heterogeneity of open-pollinated populations contrasts with the homogeneity (or virtually so) of inbred lines, clones and hybrids.
Population improvement methods fall naturally into two groups, those based on purely phenotypic selection, normally called mass selection, and those based on selection with progeny testing. Interpopulation improvement utilizes the concept of open breeding populations; allowing genes for flow from one population to another. Plants in one population (cultivar, strain, ecotype, or any germplasm source) are crossed either naturally (e.g., by wind) or by hand or by bees (commonly Apis mellifera L. or Megachile rotundata F.) with plants from other populations Selection is applied to improve one (or sometimes both) population(s) by isolating plants with desirable traits from both sources.
There are basically two primary methods of open-pollinated population improvement. First, there is the situation in which a population is changed en masse by a chosen selection procedure. The outcome is an improved population which is indefinitely propagable by random-mating within itself in isolation. Second, the synthetic variety attains the same end result as population improvement but is not itself propagable as such; it has to be reconstructed from parental lines or clones. These plant breeding procedures for improving open-pollinated populations are well known to those skilled in the art and comprehensive reviews of breeding procedures routinely used for improving cross-pollinated plants are provided in numerous texts and articles, including: Allard, Principles of Plant Breeding, John Wiley and Sons, Inc. (1960); Simmonds, Principles of Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda, Quantitative Genetics in Maize Breeding, Iowa State University Press (1981); and, Jensen, Plant Breeding Methodology, John Wiley and Sons, Inc. (1988). Detailed breeding methodologies specifically applicable to alfalfa are provided in Alfalfa and Alfalfa Improvement, supra.
2. Mass Selection
In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and their is no control over pollination, mass selection amounts to a form of random mating with selection. As stated above, the purpose of mass selection is to increase the proportion of superior genotypes in the population.
3. Synthetics
A synthetic variety is produced by crossing inter se a number of genotypes selected for good combining ability in all possible hybrid combinations, with subsequent maintenance of the variety by open pollination. Whether parents are (more or less inbred) seed-propagated lines, as in some sugar beet and beans (Vicia) or clones, as in herbage grasses, clovers and alfalfa, makes no difference in principle. Parents are selected on general combining ability, sometimes by test crosses or topcrosses, more generally by polycrosses. Parental seed lines may be deliberately inbred (e.g. by selfing or sib crossing). However, even if the parents are not deliberately inbred, selection within lines during line maintenance will ensure that some inbreeding occurs. Clonal parents will, of course, remain unchanged and highly heterozygous.
Whether a synthetic can go straight from the parental seed production plot to the farmer or must first undergo one or two cycles of multiplication depends on seed production and the scale of demand for seed. In practice, grasses and clovers are generally multiplied once or twice and are thus considerably removed from the original synthetic.
While mass selection is sometimes used, progeny testing is generally preferred for polycrosses, because of their operational simplicity and obvious relevance to the objective, namely exploitation of general combining ability in a synthetic.
The number of parental lines or clones that enter a synthetic vary widely. In practice, numbers of parental lines range from 10 to several hundred, with 100-200 being the average. Broad based synthetics formed from 100 or more clones would be expected to be more stable during seed multiplication than narrow based synthetics.
Synthetics in alfalfa are used in advanced generations as commercial cultivars. The parents are always selected for some particular trait or traits but seldom for combining ability per se. Synthetic cultivars permit the expression of heterosis to a degree, usually less than hybrids, while providing a practical method for seed multiplication.
Parents for synthetic cultivars in alfalfa are selected by many different methods. In an open breeding system the parents can be selected from such diverse sources as ecotypes, cultivars, and experimental strains. Although production of a synthetic cultivar is relatively simple, a wise choice of parents for the Syn 0 generation is crucial, for this will determine the performance of the synthetic. Decisions as to which and how many parents to include, fix the minimum degree of inbreeding that the eventual cultivar will sustain in subsequent generations.
4. Hybrids
A hybrid is an individual plant resulting from a cross between parents of differing genotypes. Commercial hybrids are now used extensively in many crops, including corn (maize), sorghum, sugarbeet, sunflower and broccoli. Hybrids can be formed a number of different ways, including by crossing two parents directly (single cross hybrids), by crossing a single cross hybrid with another parent (three-way or triple cross hybrids), or by crossing two different hybrids (four-way or double cross hybrids).
Strictly speaking, most individuals in an outbreeding (i.e., open-pollinated) population are hybrids, but the term is usually reserved for cases in which the parents are individuals whose genomes are sufficiently distinct for them to be recognized as different species or subspecies. Hybrids may be fertile or sterile depending on qualitative and/or quantitative differences in the genomes of the two parents. Heterosis, or hybrid vigor, is usually associated with increased heterozygosity which results in increased vigor of growth, survival, and fertility of hybrids as compared with the parental lines which were used to form the hybrid. Maximum heterosis is usually achieved by crossing two genetically different, highly inbred lines.
Theoretically, maximum heterozygosity and hybrid vigor in alfalfa is believed to be expressed only in a tetra-allelic condition (Bingham et al., Maximizing heterozygosity in authopolyploids 471-489, In Polyploidy: Biological Relevance, Lewis (ed.), Plenum Press (1979); Bingham et al., Maximizing Heterozygosity in Autopolyploids 130-143, In Better Crops for Food, CIBA Found. Symp. 97, Pitman Books (1983)). This could be accomplished by using four parental strains instead of the two proposed for the production of most diploid hybrid cultivars. In this way up to 75% of the plants in commercial alfalfa plantings could be double cross hybrids and thus potentially tetra-allelic. On a practical basis, hybrid alfalfa has not been commercially viable because: 1) the cost of seed production is prohibitive due to the fact that seed is harvested only from the female plants, not all of the plants in the production field; and, 2) the benefits of hybrid yield, such as increased forage yield due to hybrid vigor, have not been sufficient to offset the increased costs of producing the hybrid alfalfa seed.
The production of hybrids is a well-developed industry, involving the isolated production of both the parental lines and the hybrids which result from crossing those lines. For a detailed discussion of the hybrid production process, see, e.g., Wright, Commercial Hybrid Seed Production 8:161-176, In Hybridization of Corp Plants, supra
1. Introduction
To introduce a desired gene or set of genes by conventional methods requires a sexual cross between two lines, and then repeated back-crossing between hybrid offspring and one of the parents until a plant with the desired characteristics is obtained. This process, however, is restricted to plants that can sexually hybridize, and genes in addition to the desired gene will be transferred.
Recombinant DNA techniques allow plant researchers to circumvent these limitations by enabling plant geneticists to identify and clone specific genes for desirable traits, such as resistance to an insect pest, and to introduce these genes into already useful varieties of plants. Once the foreign genes have been introduced into a plant, that plant can than be used in conventional plant breeding schemes (e.g., pedigree breeding, single-seed-descent breeding schemes, reciprocal recurrent selection) to produce progeny which also contain the gene of interest.
Genes can be introduced in a site directed fashion using homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome.
Homologous recombination and site-directed integration in plants are discussed in U.S. Pat. Nos. 5,451,513, 5,501,967 and 5,527,695.
2. Transformation Methods
Methods of producing transgenic plants are well known to those of ordinary skill in the art. Transgenic plants can now be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; viral-mediated transformation; and Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos. 5,405,765, 5,472,869, 5,538,877, 5,538,880, 5,550,318, 5,641,664, 5,736,369 and 5,736369; Watson et al., Recombinant DNA, Scientific American Books (1992); Hinchee et al., Bio/Tech. 6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama et al., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839 (1990); Mullins et al., Bio/Tech. 8:833-839 (1990); and, Raineri et al., Bio/Tech. 8:33-38 (1990)).
Transgenic alfalfa plants have been produced by many of these methods including, but not limited to, agrobacterium-mediated transformation (Wang et al., Australian Journal of plant Physiology 23(3):265-270 (1996); Hoffman et al., Molecular Plant-Microbe Interactions 10(3):307-315 (1997); Trieu et al., Plant Cell Reports 16:6-11 (1996)) and particle acceleration (U.S. Pat. No.5,324,646).
Transformation has also been successfully accomplished in clover using agrobacterium-mediated transformation (Voisey et al., Biocontrol Science and Technology 4(4):475-481 (1994); Quesbenberry et al., Crop Science 36(4):1045-1048(1996); Khan et al., Plant Physiology 105(1):81-88 (1994); Voisey et al., Plant Cell Reports 13(6):309-314 (1994)).
3. Transgenes
Genes successfully introduced into plants using recombinant DNA methodologies include, but are not limited to, those coding for the following traits: seed storage proteins, including modified 7S legume seed storage proteins (U.S. Pat. Nos. 5,508,468, 5,559,223 and 5,576,203); herbicide tolerance or resistance (U.S. Pat. Nos. 5,498,544 and 5,554,798; Powell et al., Science 232:738-743 (1986); Kaniewski et al., Bio/Tech. 8:750-754 (1990); Day et al., Proc. Natl. Acad. Sci. USA 88:6721-6725 (1991)); phytase (U.S. Pat. No. 5,593,963); resistance to bacterial, fungal, nematode and insect pests, including resistance to the lepidoptera insects conferred by the Bt gene (U.S. Pat. Nos. 5,597,945 and 5,597,946; Hilder et al., Nature 330:160-163; Johnson et al., Proc. Natl. Acad. Sci. USA, 86:9871-9875 (1989); Perlak et al., Bio/Tech. 8:939-943 (1990)); lectins (U.S. Pat. No. 5,276,269); and flower color (Meyer et al., Nature 330:677-678 (1987); Napoli et al., Plant Cell 2:279-289 (1990); van der Krol et al., Plant Cell 2:291-299 (1990)).
Transgenic alfalfa plants have been produced using a number of different genes isolated from both alfalfa and non-alfalfa species including, but not limited to, the following: the promoter of an early nodulin gene fused to the reporter gene gusA (Bauer et al., The Plant Journal 10(1):91-105 (1996); the early nodulin gene (Charon et al, Proc. Natl. Acad. of Sci. USA 94(16):8901-8906 (1997); Bauer et al., Molecular Plant-Microbe Interactions 10(1):39-49 (1997)); NADH-dependent glutamate synthase (Gantt, The Plant Journal 8(3):345-358 (1995)); promoter-gusA fusions for each of three lectin genes (Bauchrowitz et al., The Plant Journal 9(1):31-43 (1996)); the luciferase enzyme of the marine soft coral Renilla reniforms fused to the CaMV promoter (Mayerhofer et al., The Plant Journal 7(6):1031-1038 (1995)); Mn-superoxide dismutase cDNA (McKersie et al., Plant Physiology 111(4): 1177-1181 (1996)); synthetic cryIC genes encoding a Bacillus thuringiensis delta-endotoxin (Strizhov et al., Proc. Natl. Acad. Sci. USA 93(26):15012-15017 (1996)); and glucanse (Dixon et al., Gene 179(1):61-71 (1996); Masoud et al., Transgenic Research 5(5):313-323)). Genes of particular interest to alfalfa breeding and production include those controlling the following traits:
1. Herbicide resistance: Roundup(copyright), Arsenal(copyright), and sulfonyl urea herbicides such as chlorsulfuron.
2. Insect resistance: Use of Bt or other genes conferring resistance to important insect pests such as Lygus (Lygus sp.), alfalfa caterpillar (Colias sp.), and alfalfa weevil (Hypera sp.).
3. Forage quality: Increasing rumen escape (bypass) protein, which would improve feed efficiency and utilization; decreased lignin content, which would improve digestibility and animal performance.
4. Physiological: Elimination of leaf senescence, would improve yield and quality as well as decrease leaf disease development (see, U.S. Pat. No. 5,689,042).
Genes successfully transferred into clover using recombinant DNA technologies include, but are not limited to, the following: Bt genes (Voisey et al., supra); neomycin phosphotransferase II (Quesbenberry et al., supra); the pea lectin gene (Diaz et al., Plant Physiology 109(4):1167-1177 (1995); Eijsden et al., Plant Molecular Biology 29(3):431-439 (1995)); the auxin-responsive promoter GH3 (Larkin et al., Transgenic Research 5(5):325-335 (1996); seed albumin gene from sunflowers (Khan et al., Transgenic Research 5(3):179-185 (1996)); and genes encoding the enzymes phosphinothricin acetyl transferase, beta-glucuronidase (GUS) coding for resistance to the Basta(copyright) herbicide, neomycin phosphotransferase, and an alpha-amylase inhibitor (Khan et al., supra).
These prior art methods of producing hybrid crop plants or individual plants either eliminate/maintain certain genotypes of plants entirely by spraying a particular herbicide, or they utilize a combination of male/female sterility/fertility genes and herbicide resistance genes to select for certain genotypes.
There is currently a need for methods of maintaining as many transgenic individuals as possible in an open pollinated or synthetic population. Thus, there is currently a need for methods of concentrating a transgene in a heterozygotic population such that all of the other genes, except the transgene, are maintained in the heterozygotic condition. Such methods would allow a shifting of an open pollinated or synthetic population to one that contains a higher proportion of a desired transgene which had been introduced into such populations. In this way, as many heterozygotic individuals as possible would express the transgene in such an open pollinated or synthetic population.
The capability of the present method to bias a heterozygous phytotoxin resistant plant towards producing seeds carrying the phytotoxin resistance gene will now be exemplified by the following non-limiting experiments.